BioMed Research International / 2013 / Article / Fig 3

Clinical Study

A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

Figure 3

Cell migration in scratch and Boyden chamber assays. (a) Mitomycin-C (MMC) inhibition of SMC proliferation. SMC treated with or without MMC (40 μg/mL for 30 min at 37°C) were plated in 6-well plates and subsequently harvested. Cell counts were manually performed on a hemocytometer after 1 h, 24 h, and 48 h; cell counts are expressed as the mean ± one standard deviation of triplicates. Trypan blue exclusion showed no differences in cell viability over 48 hr ( %). MMC treatment leads to stable numbers of cells collected at 24 h and 48 h with significantly increased proliferation in the non-MMC-treated populations ( ). (b) Scratch assay; no differences are seen in the extent of migration between cells cultured in no chemokine versus 25–100 ng/mL PDGF; over the 3–6 hours of this assay, no significant difference is seen between control cells and those treated with MMC. (c) Transwell assay; different concentrations of PDGF were added to the lower chamber of transwell devices at time zero; cells on the lower aspect of the transwell insert were enumerated after 6 h, 12 h, or 24 h. Compared with no PDGF in the lower chamber, there were significantly greater cell numbers of migrated cells in 10 ng/mL, 25 ng/mL, and 50 ng/mL PDGF group after 12 h ( ). After 24 hours, migrated cell numbers in transwells with 5–50 ng/mL PDGF were not significantly different relative to chambers without PDGF. After 24 hours, migrated cell numbers when 100 ng/mL PDGF was present in the lower chamber were significantly reduced relative to the control 0 ng/mL PDGF group. (d) Chemokinesis in transwells; the same 50 ng/mL concentration of PDGF was loaded into bottom chamber, top chamber, or both bottom and top chambers; cell migration was analyzed after 6 h, 12 h, or 24 h. Relative to no PDGF in either chamber, cell migration was significantly greater with 50 ng/mL in the lower chamber at 12 hours ( ); by 24 hours, there was no significant difference between control chambers and 50 ng/mL PDGF. Notably, adding PDGF to the upper chamber, with or without PDGF in the lower chamber, led to significantly increased migration at 12 hours relative to PDGF in the lower chamber alone ( ).

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